The benefit of anti-angiogenic therapy in EGFR exon 21 L858R mutant non-small cell lung cancer patients: a retrospective study

Patients with epidermal growth factor receptor (EGFR) exon 21 L858R substitution benefit less from standard EGFR tyrosine kinase inhibitor (TKI) treatment, and whether anti-angiogenic therapy was beneficial to the EGFR L858R subpopulation was inconclusive. A retrospective study was conducted to investigate the survival benefit and the target characteristics of the anti-angiogenic agent in the EGFR L858R patients in our center, comparing those treated with or without anti-angiogenic therapy (cohort A and cohort B). At the median follow-up time of 31.0 months vs 32.7 months (cohort A vs. B) respectively, Cohort A (n = 58) had a significantly prolonged median OS compared to Cohort B (n = 101) (60.0 months vs.37.0 months, HR 0.51, p = 0.016). Anti-angiogenic therapy significantly prolonged the OS in patients with liver metastases (NA vs.26.0 months, HR 0.17, p = 0.023) comparing to patients without liver metastases (60.0 months vs.37.0 months, HR 0.63, p = 0.129). For brain metastatic patients, anti-angiogenic treatment tended to improve median OS with (65.0 months vs.35.0 months, HR 0.29, p = 0.068) or without brain radiotherapy (73.0 months vs.29.0 months, HR 0.24, p = 0.171). The grade 3 or more adverse events were manageable and consistent with previous studies. Patients with EGFR L858R mutation treated with anti-angiogenic therapy in their course of treatment had a significantly prolonged OS compared to those who had never received an anti-angiogenic agent. Patients with liver metastases might benefit more from anti-angiogenic therapy than those without.

. However, the recent survival updates of both studies failed to reveal any significant impact on overall survival (OS), despite numerical advantages 16 . The RELAY study and the most recent ARTEMIS-CTONG1509 study had a similar result with PFS in the L858R subgroup, but the OS data of both trials remain immature to date 17,18 . Notably, bevacizumab treatment beyond progression with chemotherapy has been investigated in a randomized phase 3 Avastin in All Lines Lung (AvaALL) trial in NSCLC 19 . Data from the AvaALL study indicated that there might be some advantages from treatment with bevacizumab across multiple lines. The REVEL study tested the addition of ramucirumab to second-line docetaxel treatment. Patients with previous bevacizumab treatment were enrolled in the study, significant PFS and OS were both reached 20 , indicating anti-angiogenic therapy may have a benefit after progression. However, the benefit of anti-angiogenic therapy beyond anti-angiogenesis plus EGFR-TKI (A + T) resistance had not been shown in a clinical study, though frequently seen in real-world practice. Based on previous evidence, and a lack of data for the benefit of anti-angiogenic agents used across multiple lines of treatments in the EGFR L858R mutant patients, we conducted a retrospective study that sought to investigate the survival benefit and the target characteristics of the anti-angiogenic agent in the mentioned population.

Results
Clinical characteristics. A total of 159 patients with baseline EGFR exon 21 L858R mutated NSCLC met the study's inclusion criteria were enrolled in the study at our center (Cancer center, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China) between Jan 1, 2015, to Jan 1, 2021. 58 patients received anti-angiogenic therapy during their course of treatment. 101 patients never received any systemic antiangiogenic agent. Baseline patient clinical characteristics are listed in Table 1, including age, gender, smoking history, ECOG score, Stage (III/IV), histological type, concurrent mutations, brain metastases, liver metastases, and TKI received. There were no differences in clinical characteristics between patients treated with or without anti-angiogenic therapy (cohort A and cohort B). For patients in cohort A who had received anti-angiogenic therapy, 16 patients received A + T in the first line, 12 in the second line, 5 in the third line, and 1 in the fourth line, respectively. The others received anti-angiogenic agents after standard EGFR TKI treatment. Seven patients received crossline anti-angiogenic therapy after progression. For the variety of anti-angiogenic agents, 42 patients received bevacizumab, 14 received apatinib, and 11 received anlotinib. Patients may receive more than one anti-angiogenic agent during their treatment course. Of the 23 patients who had concurrent mutations in cohort A and the other 23 in cohort B. One had 1st line T790M, 20 had 2nd line T790M, 1 had 1st line TP53, and 1 had 1st line BRAF mutation in cohort A; Four had 1st line T790M, 14 had 2nd line T790M, 1 had 1st line S768I, 1 had 1st line L861Q, 1 had 1st line PI3K, 1 had 2nd line MET, and 1 had 1st line HER2 in cohort B.
Overall survival analyses in patients with liver metastasis. The OS of patients treated with or without anti-angiogenic therapy by the status of baseline liver metastases was analyzed. OS was significantly improved in Cohort A compared to Cohort B (NA vs.26.0 months, 95% CI NA-NA for cohort A, 95% CI 8.9-43.1 months for cohort B, HR 0.17, 95% CI 0.04-0.78, p = 0.023), while the comparison without liver metastasis was not significant (60.0 months vs.37.0 months, 95% CI 30.3-89.7 months for cohort A, 95% CI 26.9-47.1 months for cohort B, HR 0.63, 95% CI 0.35-1.14, p = 0.129) (Fig. 3). Patient characteristics are listed in Supplemental Table 1.
Overall survival analyses in patients with brain metastasis. For OS analyses of patients with baseline brain metastases, brain radiotherapy was used as a stratification factor, and anti-angiogenic therapy tended to benefit all patients subgroups although no statistical significance has been reached. Anti-angiogenic therapy tended to prolong median OS in patients with brain metastases who have undergone brain radiotherapy (65.0 months vs.35.0 months, 95% CI NA-NA for cohort A, 95% CI 20.8-49.2 months for cohort B, HR 0.29, 95% CI 0.08-1.10, p = 0.068), and those who have not received brain radiotherapy (73.0 months vs. 29 Table 3). The safety profile is consistent with previous reports. The adverse effects of the combination appeared to be more toxic than monotherapy, especially for hypertension and increased aminotransferase, but were mostly tolerable. Grade 3-4 hypertension was seen in 10 (17.2%) of 58 patients in cohort

Discussion
The EGFR exon 21 L858R population constitutes almost half of the sensitive mutation patients, but multiple clinical trials and meta-analyses had revealed that EGFR exon 21 L858R benefited less from EGFR-TKIs compared to exon 19 deletion regarding PFS and OS 11,21  No survival data by mutation subtype was revealed in the follow-up of the trial. Several meta-analyses had been done to confirm the rationale of this A + T combination [22][23][24] , however, there has not been a study that succeeded in showing an OS benefit for the A + T combo. The lack of an OS benefit might be explained by the prolonged survival of EGFR mutant patients. Secondly, the crossover of anti-angiogenic therapy after progression may render the first line PFS benefit effectless after several lines of combined therapy. In our study, we first tried to see if the A + T combo would prolong first-line PFS and translate into an OS benefit. Due to the limited number of cases receiving A + T (n = 16) in the first line, we did not see any statistical significance in either PFS (HR 0.90; 95% CI 0.53-1.54) or OS (HR 0.53; 95% CI 0.17-1.70) for first-line A + T. Seeing anti-angiogenic agents are used in many cases across several lines of treatments in clinical practice in  Historical data suggested patients with liver or brain metastases were especially resistant to standard EGFR TKI treatment 21,25,26 . Liver metastases represent a poor prognosis feature in NSCLC patients, and data indicating optimal systemic therapy for liver metastatic tumors has been scarce 4,26,27 . In the RELAY study, subgroup analysis suggested the addition of ramucirumab may improve PFS in patients with or without liver metastases at baseline (HR 0.48 vs 0.65) 17 . Our result reiterated the benefit of anti-angiogenic therapy in L858R patients . Kaplan-Meier survival curve of overall survival for brain metastatic patients with brain radiotherapy (A), brain metastatic patients without brain radiotherapy (B), brain metastasis-free patients (C) treated with or without anti-angiogenesis therapy.  28 . However, whether brain metastatic patients with L858R mutation were as susceptible as the others is still undetermined. Dacomitinib had the best PFS and OS data in L858R patients 8 , but patients with brain metastases were excluded from the ARCHER 1050 study, thus evidence-based data is still missing for dacomitinib in this subgroup of patients. Several meta-analyses had polled the data from the existing studies comparing A + T vs standard EGFR-TKI, benefit was seen in the A + T group despite brain metastasis 22 . Especially, in the ARTEMIS-CTONG 1509 study, bevacizumab in combination with erlotinib significantly prolonged the PFS of patients with brain metastases, from 11.1 months (95% CI 9.7-12.5) to 17 18 . However, there has not been any conclusion drawn regarding L858R mutant patients with brain metastases. In our study, we demonstrated that all patient subgroups, with or without brain metastases, and no matter the case of brain radiotherapy, all tended to fare better with anti-angiogenic therapy in their course of treatment. Generally, brain metastatic patients had a larger difference in survival, with brain radiotherapy (65.0 months vs.35.0 months; HR 0.29, 95% CI 0.08-1.   www.nature.com/scientificreports/ in the CTONG 1509 study, although OS for patients with brain metastases was numerical longer, we did not see a statistical significance. Furthermore, we considered the possibility of prolonged survival patients might have higher chances of receiving anti-angiogenic agents, leading to a selection bias resulting in longer survival in cohort A. We first did a comparison between the lines of therapy received in cohort A and cohort B. Cohort A had an average of 3.8 lines of therapy while cohort B had an average of 2.2 lines of therapy, which was significantly fewer. Thus we excluded the later-line patients treated with anti-angiogenic therapy and only compared patients treated with 1st and 2nd line A + T (cohort C) with those who were treated without anti-angiogenic therapy (cohort B) and the result showed patients in cohort C survived 23 months longer than cohort B (60.0 months vs.37.0 months, HR 0.48, p = 0.091), which was numerically the same as cohort A comparing to cohort B (60.0 months vs.37.0 months, HR 0.51, p = 0.016). However, since the number of patients in cohort C was only 26, distinctly smaller than cohort A which was 58, thus the difference was statistically insignificant (p = 0.091 vs p = 0.016). We also compared the lines of treatment in cohort C and cohort B, which was 3.0 vs 2.2. In our opinion, this discrepancy may be explained in both ways: On one hand, patients who received 1st and 2nd line anti-angiogenic therapy may genuinely have better survival and was able to receive more lines of therapy; On the other hand, lines of therapy may, in fact, be a confounding factor in this study which calls for a prospective design to overcome.
There is a multitude of reasons why tumors with L858R substitution fare worse than those with exon 19 deletion. The L858R mutation locates at the A-loop of EGFR's C-lobe, closer to the activation loop of the kinase domain. The substitution destabilizes inactive EGFR tyrosine kinase conformation and stabilizes the active conformation. TKIs generally had a lower affinity with L858R than 19 deletions, hence lower sensitivity 29 . Differences in the phosphorylation of downstream ERK and AKT pathways were also observed 30 . There were reports showing tyrosine 845 is highly phosphorylated in the L858R mutant, leading to an upregulation of the STAT3 cell survival pathway 31 . Interestingly, the L858R mutant also tends to have a poorer immune profile, represented by a higher tumor mutation burden and upregulation of the CXCL12-CXCR4 chemokine receptor pathway 32,33 . However, whether an interaction between L858R tyrosine kinase activation and the VEGFR pathway exists or not remains to be elucidated.
Based on previous trial data, there are a few drawbacks to the A + T combination. One of them is the T790M positive rate. In the ARTEMIS trial, patients from the combination-arm developed fewer acquired T790M resistance when progressed (41% in combination vs 61% in monotherapy) 18 . The same trend was observed in the RELAY trial, with respectively 25% vs 30% T790M mutation for the combination and monotherapy groups 17 . This loss of second-line breaches may be countered by saving anti-angiogenic agent for later lines or combining it with 3rd generation TKI, but trials to answer this specific question are still ongoing.
The present study has a few limitations. First, this was a retrospective design study from a single center. Ideally, it would be better to control the anti-angiogenic treatment in just the first and second line, and evaluate PFS1, PFS2, and OS, respectively, but limited by the numbered of cases receiving A + T in each line, we were unable to independently evaluate the addition of anti-angiogenic treatment in each line of therapy. Second, since we aim to investigate anti-angiogenic in not only L858R patients receiving first-line treatment but also at later lines, thus patients who survived longer may have a better chance to receive therapy, However, we tried to limit this shortcoming by doing an analysis excluding patients receiving later-line anti-angiogenic agent. The result echoed our initial finding that L858R mutant patients might benefit from anti-angiogenic therapy. Even though this was a real-world study with limitations we have uncovered clues for the benefit of anti-angiogenic therapy across all lines of treatment and underlined its possible target patients. The questions of when and how and to whom with EGFR L858R substitution should we add the anti-angiogenic agent are still inconclusive, thus warranting more efforts for the benefit of this specific mutation population.
In conclusion, this study demonstrated that NSCLC patients who harbored L858R substitution treated with anti-angiogenic therapy have better overall survival, especially for those with liver or brain metastasis.

Patients and methods
Patients. All NSCLC patients with EGFR exon 21 L858R substitution who received first-line EGFR-TKI were enrolled in this study for survival analysis. The main inclusion criteria were as follows: unresectable stage III/IV (according to the 8th American Joint Committee on Cancer Staging System), histologically confirmed non-squamous NSCLC with EGFR exon 21 L858R mutation, at least one radiological response evaluation according to Response Evaluation Criteria in Solid Tumors (RECIST) V1.1, treated with first-line EGFR-TKI, aged over 18 years old. The main exclusion criteria included previous EGFR-TKI treatment, adjuvant use of EGFR-TKI, and diagnosed concomitantly with other cancers besides NSCLC.
Ethics statement. This is a retrospective real-world study approved by the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine Ethics Committee (20211215-31) and conducted according to the principles of the Declaration of Helsinki. The requirement for informed consent was waived by the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine Ethics Committee in view of the retrospective nature of the study.

Study design.
Clinical information was collected, including but not limited to demographic information, Smoking history, ECOG score, histological types, stages, metastases sites, current or subsequent mutations, anti-angiogenic treatments, 1st, 2nd, and 3rd line TKIs, chemotherapy information, radiotherapy information, immunotherapies, PFS, OS, and adverse events. The OS of patients receiving anti-angiogenic agents or not throughout the treatment course was compared, and stratified by patients with or without liver metastases or brain metastases.